CHM161L Biochemistry 2 Laboratory 2nd Quarter SY 2013-2014

Crude Protein Determination of Monde Nissin Wafer Using Bradford Assay Austria, Meynarda, Arceo, Chrissieb

ABSTRACT The experiment determined the crude protein content of a Monde Nissin Wafer sample. The determination of crude protein is important in the quality and labelling of a food product. This also gives consumers an idea of how much protein is in their goods. The glassware used were crucibles and covers, filter paper, desiccator, forced draft oven, and a muffle furnace. The sample for crude protein determination was acid digested, filtered and ran in the ultraviolet visible spectrophotometer. Included in the results and discussions are the plot of absorbance vs concentration of the standard that was ran at 595 nm, which is the wavelength at which protein can be detected in the dye used.

Introduction All cells have proteins as a component abundant to it. Almost all proteins are important for biological functions and cell structure except for storage proteins because they serve as metal ions and amino acids reserves. The proteins found in food have very complex structures that have been purified and characterized by researchers. Proteins are composed of elements including hydrogen, carbon, nitrogen, oxygen and sulfur. The building blocks of proteins are -amino acids, and the links that connect amino acid residues in a protein are called peptide bonds. Bradford assay was the method used in determining the crude protein content of a Monde Nissin Wafer sample. This involved the binding of Coomassie Brilliant Blue G-250 to the protein as described by Bradford in 1976. A shift in the absorption maximum of the dye from 465 to 595 nm was observed as the dye was bound to the protein. The dye binding process was completed in approximately 2 minutes with good color stability for 1 hour. The sample was acid digested to avoid interference from cations such as sodium or potassium and from carbohydrates such as sucrose. The objective of this experiment was to determine the crude protein content of a Monde Nissin Wafer sample. Determining the crude protein content of a food sample is important because it is used in the

labeling and nutrition information and for quality control of a product. This also includes pricing, functional property investigation, and biological activity determination. Beakers and a UV-Vis spectrophotometer were used in the experiment. The reagents used for the acid digestion were 1.25% sulfuric acid and 2.50% NaOH. The wavelength absorbance used was at 595nm. Methodology The materials used were a UV-Vis spectrophotometer, volumetric flasks, beakers, graduated cylinders, pipets, micro pipets, suction bulbs, and stirring rods. Protein standards were prepared in the same buffer as the samples to be assayed. A convenient standard curve was made using bovine serum albumin (BSA) with concentrations of 0, 250, 500, 1000, 1500, 2000 g/mL for the standard assay, and 0, 10, 20, 30, 40, 50 g/mL for the microassay. The standard solutions to be subjected to the UV-Vis contained 22.5mL water, 1.5mL standard, and 6mL Bradford reagent. The standards were made by first making a mother solution of 100mL of 10mg/mL BSA solution. The following standards were created using computation with the formula, and then diluted to mark. For the sample, two (2) grams of sample was measured and then diluted with 80mL distilled

Professor, School of Chemical Engineering and Chemistry, bStudent, CHM161L/C21

CHM161L Biochemistry 2 Laboratory 2nd Quarter SY 2013-2014

water. About 1.5mL of the solution was then mixed with 22.5mL water with 6mL Bradford reagent. The blank on the other hand was a mixture of 24mL water and 6mL Bradford, which was the first thing to be subjected to the UV-Vis, followed by the standard, and then the sample itself under an absorbance of 595nm. Results and Discussion

Table 1 Standards and Sample Absorbance Reading Concentration of standard at 595nm 0 250 500 Absorbance Sample reading % Protein

0 0.07 0.0946 0.1677 0.2532 0.3122 0.1496170. 117678

0.2176 0.2264 0.2407

6.70 6.99 7.47

Absorbance vs Concentration
0.35 0.3 Absorbance, nm 0.25 0.2 0.15 0.1 0.05 0 0 1000 2000 3000 Concentration of Standard, ppm Linear (Series1) y = 0.0002x + 0.0166 Series1

1000 1500 2000 AverageStan dard Deviation

0.27823 30.053 897

7.053333 0.38888 87

Conclusion Figure 1 Graph of Absorbance Reading at 595nm. The data shows the absorbance reading of the sample that was subjected to a UV-Vis spectrophotometer. The true value of the protein was not included in the nutritional information of the Monde Nissin Wafer, so there is no way that we can know if we got a correct value for the percentage of protein. Although relating the values obtained to the ones recorded by Mamat et al (2010) that ranged from 6.2 to 7.0 percentage protein, are relatively close. The objectives of the experiment which were to determine the crude protein content of Monde Nissin Wafer were met. The percentage of crude protein that was determined was just relatively close to the ones in the literature. The sources of error might come from the interferences that were present during subjection to UV-Vis. Another source of error may come from the standards used. The reagent may have impurities when it was used.